Prevention and/or treatment of allergic conditions

ABSTRACT

The present invention relates to the use of an inhibitor of cysteine proteinase activity in conjunction with an inhibitor of any serine proteinase activity other than trypsin for the manufacture of a medicament for the prevention or treatment of a condition in which an allergen traverses an epithelial barrier such as asthma. Also included in the invention are formulations and kits containing serine and cysteine proteinase inhibitors and their use in the prevention or treatment of conditions in which an allergen traverses an epithelial barrier.

This application is a 371 of PCT/GB98/03721, filed Dec. 11, 1998.

The present invention relates to the prevention and/or treatment ofallergic conditions and more particularly to such conditions in which apotential allergen must traverse an epithelial barrier. The inventionhas particular, but not sole, application to the prevention and/ortreatment of asthma.

Asthma is the most common chronic disease of childhood and a majordebilitating and life threatening condition. It is characterised byacute hypersensitivity, chronic bronchial reactivity and damage anddisruption to lung epithelium.

A primary risk factor for asthma is the sensitisation of the lung toairborne allergens such as proteins excreted in the faecal pellets ofhouse dust mites (HDM) belonging to the genus Dermatophagoides (e.g. Dpteronyssinus, D. Farinae). When inhaled, HDM faecal pellets impact uponthe fluid-covered epithelial surface of large diameter airways. Theresulting hydration of HDM faecal pellets will trigger a rapid and totaldischarge of the major allergenic proteins thus achieving a high localconcentration of HDM proteins on the airway lining. Sensitisationinvolves allergen detection by antigen presenting cells which arenormally protected from the environment by the lung epithelium. Themechanism by which the allergens are able to traverse the epithelialbarrier is not fully understood.

There is now an increasing body of evidence that several major allergensfrom HDMs exhibit catalytic competence as enzymes and this has promptedsuggestions that these enzymatic actions might be important in allergicsensitisation and to the perpetuation of established allergicinflammatory reactions.

Most data concerning proteinase activity in mite allergens currentlyrelate to those of groups 1, 3, 6 and 9 from mites of the genusDermatophagoides. The group 1 allergens are cysteine proteinases andhave been the subject of greatest scrutiny whereas a lesser amount ofinformation exists concerning the enzymatic effects of the group 3,group 6 and group 9 allergens which share sequence identity witharchetypal serine proteinases and which are themselves catalyticallycompetent.

It has been proposed that the cysteine proteinase activity is importantin exacerbating the allergic response in asthma because it cleaves CD23,a low affinity IgE receptor, on the surface of antibody producing cells.Cleavage results in positive feedback that causes an increase in igEsecretion, thus augmenting the allergic response. On this basis,WO-A-97/04004 (Peptide Therapeutics) proposes that inhibitors ofcysteine proteinases may be used inter alia for asthma prophylaxis.However we do not believe that the mechanism proposed in WO-A-97/04004is likely to operate in vivo. The reason for this is that allexperiments on cleavage of CD23 have been carried out on cells inculture and the concentrations of Der p 1 required to produce cleavagewere in our view unrealistically large. The cells concerned would, invivo, be located in the tissues or the blood. It is, in our view,extremely unlikely that allergen concentrations would reach the levelsrequired to produce CD23 cleavage on these locations. There is noevidence that they do, nor that CD23 cleavage takes place in vivo.

Kalsheker N. et al. (1996) Biochem. Biophys. Res. Comms. (USA) 221/1pages 59-61 discloses that the serine proteinase α₁-antitrypsin protectsthe lower respiratory tract from damage by proteinases released in thelung during inflammation. The cysteine proteinase Der p1 is shown tocleave the proposed reactive loop of the serine proteinase inhibitorα₁-antitrypsin and this mechanism is proposed as being important in thepathogenesis of asthma. Also disclosed is that α₁-antitrypsin deficiencyis linked to the incidence of childhood asthma. However there is nodisclosure of how allergic conditions (such as asthma) in which anallergen must traverse an epithelial barrier may be treated orprevented.

Stewart G. et al. (1991)Int. Arch. Allergy Appl. Immunol. 95/2-3 pages248-256 discloses that dust mite faeces contains three serineproteinases and a cysteine proteinase along with various other enzymes.It also discloses that the cysteine proteinase and at least one of theserine proteinases are allergenic. However there is no disclosure of howallergic conditions such as asthma in which an allergen must traverse anepithelial barrier may be treated or prevented.

In spite of the considerable effort which has taken place in the fieldof asthma research, there remains a need for improved methods for theprevention and/or treatment of asthma (and other allergic conditions inwhich a potential allergen most traverse an epithelial barrier).

In its first, broadest aspect the invention provides for the preventionand/or treatment of allergic conditions (of the type in which apotential allergen must traverse an epithelial barrier) by theinhibition of cysteine proteinase activity and serine proteinaseactivity.

The invention is applicable particularly (but not exclusively) to thetreatment of asthma for which the cysteine proteinase activity to beinhibited is preferably that of Der p 1 whereas the serine proteinaseactivity to be inhibited may be that of any serine proteinase other thantrypsin, preferably an allergen serine proteinase and more preferablyDer p 3, Der p 6 and/or Der p 9.

The invention has been based on our experimental studies (set out inmore detail below) which have demonstrated that the key initial step inallergic sensitisation to house dust mite allergens is mediated by bothcysteine and serine proteinase activity. We have found that thisactivity causes disruption of tight junctions between the cells of theepithelium thus increasing epithelial permeability and permitting theallergen to traverse the epithelium. By this means, the allergens maygain access to, and interact with, dendritic antigen presenting cells toproduce an allergic response. Cysteine proteinase inhibitors inhibit thecysteine proteinase allergens but not the serine proteinase allergensand so do not completely block tight junction breakdown. Similarly,serine proteinase inhibitors block the effects of serine proteinaseallergens but not the cysteine proteinase allergens and so do notcompletely block tight junction breakdown. Partial inhibition wouldstill allow an allergic response to be produced. Inhibition of both thecysteine and serine proteinase activity of the allergens is necessary toinhibit disruption of the tight junctions completely and thus thegeneration of an allergic response.

Although the invention is applicable particularly to the preventionand/or treatment of asthma it may be applied to a range of otherallergic conditions including rhinitis, allergic conjunctivitis, atopicdermatitis and food allergies.

The treatment and/or prevention of the allergic condition may beeffected by means of

(i) a formulation (which provides a second aspect of the invention)having cysteine and serine proteinase inhibitory activity; or

(ii) a kit (which provides a third aspect of the invention) comprisingan inhibitor of cysteine proteinase activity and an inhibitor of serineproteinase activity.

In the formulation of the second aspect of the invention, a singleinhibitor compound may provide the required inhibition of serine andcysteine protease activity. However, more usually, and in accordancewith a preferred embodiment of the second aspect of the invention, theinhibition of cysteine proteinase activity and serine proteinaseactivity will be provided by separate inhibitor compounds.

Where separate inhibitory compounds are used as in the kit of the thirdaspect of the invention, they may be used simultaneously with each otheror sequentially.

If necessary more than one type of cysteine proteinase activity and/ormore than one type of serine protease activity may be used to providethe required spectrum of activity.

The invention is applicable principally (but not exclusively) totherapeutic treatments.

Therefore according to a fourth aspect of the invention there isprovided the use of an inhibitor of cysteine proteinase activity inconjunction with an inhibitor of any serine proteinase activity otherthan trypsin for the manufacture of a medicament for the prevention ortreatment of a condition in which an allergen traverses an epithelialbarrier.

According to a fifth aspect of the invention there is provided a methodof treating a subject for the prevention or treatment of a condition inwhich an allergen traverses an epithelial barrier comprisingadministering to the subject therapeutically effective amount(s) of aninhibitor of cysteine proteinase activity and an inhibitor of serineproteinase activity.

These therapeutic treatments may be effected using the composition ofthe second aspect of the invention or the kit of the third aspect of theinvention.

The fourth and fifth aspects of the invention are particularlyapplicable to the treatment of asthma and more particularly to theprophylactic treatment thereof. By the term prophylactic treatment weinclude any treatment applied to prevent, or mitigate the effect of, asubsequent asthmatic attack. The prophylactic treatment may be given,for example, periodically to a person who is known to suffer fromasthmatic attacks with a view to preventing, or reducing the frequencyof, such attacks. Alternatively the prophylactic treatment may be givenon an ad hoc basis to a person who suffers from asthma and who is to besubjected to an environment (e.g. an allergen infected environment)which might make the onset of an asthmatic attack more likely. A furtherpossibility is for the prophylactic treatment to be given to a personwho has not developed asthma but who, for one reason or another, isbelieved to be at risk of doing so.

For the purpose of therapeutic administration, the inhibitorycompound(s) will be formulated in a pharmaceutically acceptableexcipient for delivery to the lung epithelium. Most preferably theinhibitory compound(s) will be delivered by means of an aerosol as isconventional for anti-asthmatic treatments. We do not however precludeother delivery routes.

The amount of the inhibitory compound(s) to be administered will, ofcourse, be a therapeutically effective dose. The dosage rate will dependon factors such as the weight of the patient to be treated, the severityof the asthmatic condition being treated and the activity of theinhibitors. However typical dosages will be in the range 1 to 1000microgrammes per day.

Although the invention has so far been described with particularreference to the therapeutic treatment of asthma, our finding that theinhibition of cysteine and serine proteinase activity of house dust miteallergens may be used beyond the field of therapeutic treatment. Thus,for example, it is possible to treat inanimate substrates which containor potentially contain house dust mite allergens with inhibitors inhibitcysteine and serine proteinase activity, so as to render such substrateshypoallergenic. Examples of such substrates are those which aregenerally associated with relatively high levels of allergens (e.g. softfurnishings, carpets, bedding etc.).

Examples of inhibitors of cysteine proteinase activity which may be usedfor any aspect of the invention includeL-trans-epoxysuccinyl-leucylamido-(4-guanidino)-butane (E-64) as well asthe cysteine proteinase inhibitors disclosed in WO-A-97/04004.

Examples of inhibitors of serine proteinase activity which may be usedfor any aspect of the invention include4-(2-aminoethyl)-benzenesulphonyl fluoride hydrochloride (AEBSF).

The invention will be illustrated by the following non-limiting Example(and accompanying Figures) which give results of the Example.

EXAMPLE

Using the methods described in more detail below, the Exampledemonstrates the effect on epithelial permeability of

(i) cysteine and serine proteinase fractions separated from House DustMites, and

(ii) the fractions specified under (i) in combination with inhibitors ofcysteine and serine proteinase activity.

Methods

Cell Culture

Calu-3 and MDCK cells were used as paradigms for examining intercellularjunctions of epithelia and their susceptibility to HDM proteinases andpotential inhibitors. Both cell lines express tight junctions, zonulaeadherentes and desmosomes and are thus acceptable models of celladhesion mechanisms present in the airway. Calu-3 is an adenocarcinomacell line derived from a 25-year-old Caucasian male. It has been thesubject of relatively few investigations, but is known to express tightbarrier properties on the basis of electrophysiological studies (Shen etal., 1994; Haws et al, 1994) and our own immunocytochemicalcharacterization (not shown). Cells were propagated in Eagle's minimumessential medium with Earle's salts (EMEM) supplemented with 10% v/vheat inactivated foetal calf serum (FCS), 2 mM L-glutamine,non-essential amino acids, 10μM sodium pyruvate and containing 50U/mlpenicillin and 50μg/ml streptomycin.

Madin-Darby canine kidney (MDCK) epithelial cells were cultured in EMEMcontaining 50U ml⁻¹ penicillin, 50μg ml⁻¹ streptomycin, 2 mML-glutamine, non essential amino acids and 10% v/v heat inactivated FCS.For subculture of both cell types, the cells were rinsed inphosphate-buffered saline (PBS) without calcium and magnesium and thenpartially digested using a 0.05% (w/v) trypsin and 0.02% (w/v) EDTAsolution.

All cultures were propagated at 37° C. in a humidified atmosphere of 5%carbon dioxide in air.

Coating of Transwell™ Inserts with Matrigel

Measurements of mannitol clearance were performed on confluent cellmonolayers that had been propagated on 0.4μm pore diameter CostarTranswell™ inserts coated with an ungelled ultra-thin undercoat ofMadrigel. Coating was achieved by addition of 250μl aliquots of Matrigel(diluted 1:500 v/v in EMEM) to the interior of the insert followed byambient incubation for 60 min under aseptic conditions. The solution wasthen aspirated and the inserts gently washed with medium before theaddition of a confluent density of cell suspension.

Cell Treatment Protocols and Measurement of Clearance

Cells (2-5×10⁵ per cm² growth area) were plated onto Matrigel-coatedinserts. We use the term ‘insert’ as meaning the filter unit containingthe cells and the term ‘well’ as referring to the cavities of the tissueculture plate. To monitor growth and integrity, inserts were taken atrandom, washed gently in PBS and stained under subdued illumination withacridine orange and ethidium bromide (1mg ml⁻¹ each in PBS). Insertswere examined by fluorescence microscopy and were used only whenconfluence with high viability was attained.

At confluence, the medium was aspirated from the wells and replaced withserum- and bicarbonate-free EMEM buffered with 20 mM HEPES andcontaining 2 mM L-glutamine. The medium from the inserts was then gentlyremoved and replaced with 300μl of serum-free EMEM containing[¹⁴C]-mannitol (1μCi ml⁻¹ and 1 mg ml⁻¹ unlabelled mannitol inHEPES-buffered medium). The Transwell™ plates were then equilibrated for30 min at 37° C. on a Luckham R100 orbital shaker. Triplicate 100μlaliquots of the unused labelled mannitol solution were sampled and theirradioactivity content determined by liquid scintillation spectrometry(Beckman LS6000IC) following addition of 5 ml Opti-Fluor.

After the equilibration period, the inserts were placed in fresh wellscontaining 1 ml of serum-free HEPES buffered EMEM and incubated at 37°C. with continuous gentle shaking. The medium from the original wellswas retained and its radioactivity content determined after the additionof 10 ml Opti-Fluor. These results were used to determine the tracerconcentration at time zero. At timed intervals, 20μl aliquots of thebasolateral bathing fluid were removed and the amount of ¹⁴C mannitolquantified as described above for the calculation of clearance volume.

Calculation of Epithelial Permeability

Paracellular permeability of mannitol was determined in accordance withthe procedure described in our copending U.K. Patent Application No.9715058 and was calculated from measurements of clearance volume atdefined time points. Clearance estimates were made over 3-5h and werecalculated according to the relationship: $\begin{matrix}{{V_{probe}}_{t} = {\sum\limits_{i = 1}^{l}\frac{V\quad {A_{i} \cdot {\Delta \lbrack A\rbrack}_{i}}}{( \lbrack L\rbrack )_{i}}}} & (1)\end{matrix}$

where:

V_(probe) _(t) is the clearance volume at each time point

VAi is the abluminal volume at each time point

Δ[A]i is the increase in tracer concentration between time points

[L]i is the luminal tracer concentration at each time point

Under conditions where diffusion is the sole means of transepithelialmovement of the solute, dV_(probe)/dt approximates closely to thepermeability-surface area product thus allowing estimation of thepermeability of mannitol in the composite system of cells, filter,unstirred layers and protein coating (P_(t)). Epithelial permeabilitycan be calculated from the measured variable by considering theMatrigel-coated filter and unstirred layers as a system of seriespermeabilities. Thus: $\begin{matrix}{\frac{1}{P_{l}} = {\frac{1}{P_{1}} + \frac{1}{P_{2}} + \frac{1}{P_{3}}}} & (2) \\{and} & \quad \\{\frac{1}{P_{4}} = {\frac{1}{P_{2}} + \frac{1}{P_{3}}}} & (3)\end{matrix}$

where

P₁ is the composite permeability of the system

P₁ is the component due to the epithelial cells alone

P₂ is the component due to the filter without Matrigel

P₃ is the unstirred layer component

P₄ is the permeability of the filter with the Matrigel coating

In pure diffusion systems

$\begin{matrix}{P_{3} = \frac{D}{\delta}} & (4)\end{matrix}$

where D is the free diffusion coefficient of mannitol δ is the summedthickness of unstirred layers

Unstirred layer thicknesses are independent of membrane permeabilityunder ideal conditions thus P₃ in equations (3) and (4) are identical.Subtracting equations (2) and (3) thus permits the calculation of thepermeability of the epithelial monolayer (5). $\begin{matrix}{\frac{1}{P_{l}} = {\frac{1}{P_{1}} - \frac{1}{P_{4}}}} & (5)\end{matrix}$

Analysis of variance was performed after log transformation of thepermeability data and probability values for the different treatmentsassigned using the least significant difference test. Data are presentedas the geometric mean values with indicated standard errors of nexperimental observations. Probability levels of P<0.05 were consideredstatistically significant.

Preparation of Proteinase Fractions from HDM Culture Medium

HDM proteinase allergens have not yet been prepared in catalyticallycompetent form by recombinant cellular expression of the mature enzymeprotein. For convenience, and to enable future large scale screening ofpotential inhibitors in the absence of catalytically active recombinantproteins, we sought to separate the cysteine and serine proteinaseactivity by simple biochemical fractionation of spent medium in whichHDM had been grown. During culture the HDM release allergens into themedium resulting in the accumulation of proteins suitable forpurification. Spent medium from cultures of D. pteronyssinus(Commonwealth Serum Laboratory, Parkville, Australia) was dissolved in 5volumes of phosphate buffered saline and then centrifuged at 48,400×gand 4° C. for 20 min. Ammonium sulphate was added gradually to thestirred supernatant at 4° C. to achieve a 50% saturated solution. Aftercentrifugation (48,400×g, 20 min, 4° C.) the pellet, found by enzymaticassay to be enriched in cysteine proteinase activity, was redissolved ina minimum volume of distilled water. Ammonium sulphate was added to thesupernatant from the first cut to achieve 80% saturation. The pelletresulting from further centrifugation, found to be enriched in serineproteinase activity, was resuspended in a minimum volume of distilledwater. The cysteine (50% precipitate) and serine proteinase (50-80%precipitate) fractions were separately dialysed against distilled waterovernight and then lyophilized prior to reconstitution in EMEM. Proteincontent of the extracts was measured using the Coomassie Blue techniquewith serum albumin as standard (Smith et al, 1985). Proteinase activitywas measured using the Azocoll degradation assay as described elsewhere(Herbert et al., 1995; Chavira et al., 1984). Extracts were also assayedfor the presence of endotoxin using the Limulus amebocyte lysis assay(Endotect™, ICN Biomedicals, Thame, Oxfordshire). In all cases thelevels of endotoxin were below the limit of assay detection (<0.06ngml⁻¹).

Immunoblotting of HDM Proteinase Fractions

Proteinase fractions were separated by SDS-PAGE and transferredelectrophoretically to nitrocellulose membranes. Non-specific proteinbinding was blocked with 5% w/v non-fat milk and 0.1% v/v Tween-20 inTris-buffered saline (TBS) followed by incubation with mAb 5H8 (anti-Derp 1) diluted in TBS containing 2% w/v bovine serum albumin and 0.1% v/vTween-20. Detection was by enhanced chemiluminscence technique (AmershamInternational, Buckinghamshire).

Cells were plated on 60×15 mm petri dishes and grown for 2-4 days inserum-containing EMEM under tissue culture conditions in a 5% CO₂atmosphere. Cells were then exposed to treatments in serum-free EMEMcontaining 20 mM HEPES whilst under aerobic incubation at 37° C. Atdefined time points, cells were harvested with a scraper and pooled withdetached cells in the supernatant. Cells were centrifuged at 550×g for 5min and their DNA extracted (Nucleon II, Scotlab, Coatbridge,Stratchclyde). The extracted DNA was resuspended in 100μl TE buffer (10mM Tris-HCL and 1 mM Na₂EDTA) overnight at room temperature and itspurity determined spectrophotometrically. Equal amounts of DNA wereapplied in 4:1 ratio with sample buffer (0.25% bromophenol blue and 40%w/v sucrose in water) to each lane of 2% (w/v) agarose gels andelectrophoresis performed at 50V for 2-3h in TAE buffer (0.04MTris-acetate and 0.001M EDTA). Bands of DNA in the gels (which hadethidium bromide incorporated into them) were visualized by ultra-violetlight.

The redistribution of phosphatidylserine into the outer layer of cellmembranes which occurs during the initiation of apoptosis was studiedusing annexin V staining. This was performed in 60×15 mm petri disheswhich had been modified by drilling a 1 cm diameter hole into the baseof each dish and covering the external face of this with a glasscoverslip secured in place by Sylgard (Dow Corning, Midland, Mich.,USA). A polyamide ring was mounted by means of cyanoacrylate adhesiveonto the inner face of the dish to create a glass-bottomed well whichwas then coated with an ultrathin layer of Matrigel. Cells (3×10⁴) wereplated into each well and allowed to grow for 2-3 days, after which theywere exposed to the desired experimental treatment. Following this,cells were rinsed in PBS and then in 200 ml binding buffer prior toaddition of annexin V-FITC (AV) and propidium iodide (PI) under subduedlighting conditions. After incubation for 15 min the cells were rinsedwith binding buffer and examined by fluorescence microscopy using blueand green excitation filter sets (Zeiss Axiovert 10 with oil immersionFluar objectives). Using this technique, cells in early apoptosis staingreen because FITC-conjugated annexin V binds to phosphatidylserinewhich has become reorientated into the outer leaflet of the cellmembrane (Fadok et al., 1992; Koopman et al., 1994; Vermes et al., 1995;Homburg et al., 1995). Dead cells also show red staining with PI becauseincreasing permeability of the nuclear membrane allows it to bind tonucleic acids. Photographic documentation was made using a Contax 167MTcamera and Kodak TMAX 400 film for black and white prints or Ektachrome160T for colour reversal images.

LDH activity was measured using pyruvic acid as substrate and monitoringspectrophotometrically the formation of a phenylhydrazone derivativefrom lactic acid. MDCK or Calu-3 cells were seeded onto 12-well platesand grown to confluency. Cell monolayers were exposed for 18 h to eithercontrol treatments (serum- and phenol red-free EMEM. with 0.6 mMdithiothreitol in the case of the control for the cysteine proteinasefraction) or the HDM proteinase fractions diluted in the same medium(with 0.6 mM dithiothreitol present in the cysteine proteinasefraction). At the end of the experiment the incubation medium washarvested and centrifuged at room temperature to sediment any cellswhich had detached from the wells during treatment. The firstsupernatant fraction was assayed directly for LDH activity, whereas thepellet formed by any detached cells was subjected to hypotonic lysiswith distilled water (5 min at room temperature) prior to briefcentrifugation to remove cellular debris. The resulting secondsupernatant was then assayed for LDH activity. Cells which had remainedadherent to the wells during treatment were lysed as described above andLDH activity measured. Because no significant detachment occurred duringtreatment with control media, total cellular LDH activity was defined asthat present in the lysate from adherent cells treated with serum- andphenol red free EMEM alone.

Immunocytochemical Visualization of the Effects of Inhibitors onProteinase-mediated Cleavage of Intercellular Junctions

To study the effects of proteinases and inhibitors on intercellularjunctions, MDCK cells were cultured on coverslips and treated with theappropriate proteinase and/or inhibitor for the desired time period. Thecells were fixed in ice-cold methanol before binding of rat anti-ZO-1(mAb R40.76) (Stevenson et al., 1986; Anderson et al., 1988) and mouseanti-desmoplakin (mAb 11-5F) (Parrish et al., 1987). Indirectfluorescent antibody staining was performed using FITC- andTRITC-conjugated second antibodies. Microscopy was carried out using aZeiss Axiovert microscope with x40 magnification oil immersion Fluarobjective. Specimens were illuminated using excitation and emissionfilter sets for FITC and TRITC. Cells were photographed as describedabove.

Materials

All media and cell culture reagents were purchased from ICN BiomedicalsLtd (Thame, Oxfordshire). except where stated. HBSS was obtained fromGibcoBRL, Life Technologies Ltd (Paisley). Mannitol and Triton X-100were obtained from Sigma Aldrich Ltd (Poole, Dorset) and heatinactivated foetal calf serum was from Labtech International Ltd(Uckfield, East Sussex). Matrigel was obtained from UniversalBiologicals, London. Mannitol clearance measurements were made in 12 mmdiameter Transwells with 0.4μm membrane pore size and 10μm membranethickness (Costar UK Ltd, High Wycombe, Buckinghamshire).D-[¹⁴C]-mannitol was obtained from NEN Du Pont Research Products(Stevenage, Hertfordshire), and the Opti-Fluor scintillant and thescintillation vials were from Canberra Packard Ltd (Pangbourne,Berkshire). MDCK cells were grown from stock in our laboratory. Calu-3cells were originally obtained from the American Type Culture Collection(Rockville, Md., USA) and expanded by serial passage to create a localbank of cryopreserved cells. Cells were cultured in Falcon 75 cm² cellculture flasks (Marathon Laboratory Supplies, London). Costar multiwelltissue culture plates or Transwell inserts according to the nature ofthe experiment. Agarose (molecular grade) was from Promega (Southampton,Hampshire). Assay kits for LDH measurement were purchased from Sigma;Apoalert kits were purchased from Cambridge Bioscience. Acridine orange,ethidium bromide and all other general laboratory reagents were obtainedfrom BDH (Poole, Dorset). Compound E-64(L-trans-epoxysuccinyl-leucylamido-(4-guanidino)-butane, an inhibitor ofcysteine proteinases, was obtained from Sigma. Concentrated aqueousstock solutions were stored frozen until required. The serine proteinaseinhibitor 4-(2-aminoethyl)-benzenesulphonyl fluoride hydrochloride(AEBSF) was obtained from Pentapharm, Basle, Switzerland. The matrixmetalloproteinase (MMP) inhibitor BB-250([4-(N-hydroxyamino)-2R-isobutyl-3S-(thiophen-2-yl-sulphonylmethyl)succinyl]-L-phenylalanine-N-methylamide)was provided by British Biotech Pharmaceuticals Ltd. All inhibitors weremade as concentrated stock solutions in dry Me₂SO and diluted asrequired with medium for use in experiments. Appropriate controls forthe Me₂SO vehicle were incorporated into experiments as required.Monoclonal antibody R40.76 reactive against ZO-1 was generously providedby Dr Bruce Stevenson, University of Alberta. Monoclonal Der p 1antibody 5H8 was a kind gift of Dr Martin Chapman, University ofVirginia, USA.

Results

Fractionation of Spent Mite Medium

Spent mite medium was separated into two fractions by ammonium sulphateprecipitation. The fraction yielded by precipitation with 50% ammoniumsulphate consisted of major protein bands at ˜22 KDa and 38 KDa (FIG.1a, lane 2). Tests of enzyme degradation using chromogenic substratesshowed that the catalytic activity of the 50% precipitate wasinhibitable by E-64 (not shown). Immunoblot analysis of the 50%precipitate using mAb 5H8 raised against Der p 1 revealed the presenceof a major band with an apparent mass of ˜22 KDa and a minor band at 38KDa (FIG. 1b, lane 2). In the SDS-PAGE and immunoblotting analyses thethe cysteine proteinase fraction behaved identically to Der p 1 purifiedby a combination of immunoaffinity chromatography, gel filtration andisoelectric focussing (compare lanes 1 and 2 in panels a,b of FIG. 1).Comparison of lanes 2 and 3 of the immunoblot shown in FIG. 1bdemonstrates that the 5H8 mAb also reacted with an additional range ofproteins present in the 50-80% ammonium sulphate precipitate. In theabsence of reducing agent the 50-80% ammonium sulphate precipitatefraction exhibited high catalytic activity which was attenuated byinhibitors of archetypal serine proteinases (not shown). Forconvenience, in this manuscript the 50% precipitate is subsequentlyreferred to as the cysteine proteinase fraction and the 50-80%precipitate as the serine proteinase fraction.

Effects of HDM Proteinase Fractions on Epithelial Permeability

Under the control conditions used in these experiments both MDCK andCalu-3 epithelial cells lines form tight monolayers with mannitolpermeabilities in the range 0.7-1.2×10⁻⁶ cm s⁻¹. Exposure of either MDCKor Calu-3 cell monolayers to the serine proteinase fraction produced aconcentration-related change in permeability (FIG. 2). Theconcentration-dependency of the cysteine proteinase fraction was nottested in this particular series of experiments, but the effects of purecysteine proteinase allergen Der p 1 have been previously demonstratedby us in similar in vitro models (Herbert et al., 1990; 1995).

The effects of the cysteine proteinase fraction in MDCK cell monolayerswere attenuated by the cysteine proteinase inhibitor E-64 (FIG. 3a).E-64 had no observable effect on the intrinsic permeability propertiesof the cell monolayer (FIG. 3a). The serine proteinase inhibitors AEBSFand, less effectively, SBTI both inhibited the action of the serineproteinase fraction in MDCK cells (FIG. 3b). Neither inhibitor per seexerted any observable effect on epithelial permeability (FIG. 3b). Theinhibitors were also effective when tested at the same concentration inCalu-3 cell monolayers. Calu-3 cell monolayers treated with the cysteineproteinase fraction had a mannitol permeability of (8.44±0.43)×10⁻⁶ cms⁻¹ which was reduced to (4.84±0.32)×10⁻⁶ cm s⁻¹ by E-64 (P<0.05, n=5).Calu-3 cell monolayers treated with the serine proteinase fraction had amannitol permeability of (18.1±0.02)×10⁻⁶ cm s⁻¹ which was reduced to(10.20±0.01)×10−6 cm s⁻¹ by AEBSF (P<0.05, n=5).

The class-specific proteinase inhibitors were only effective against thecognate enzyme fractions derived from the HDM cultures. FIG. 4 showsthat AEBSF, at a concentration which ablated the effects of the serineproteinase fraction, failed to inhibit the action the cysteineproteinase fraction on Calu-3 cell monolayers. Conversely, E-64 did notinhibit the permeability change caused by the serine proteinase fraction(FIG. 4).

FIG. 5 shows that treatment of MDCK cell monolayers with either thecysteine or serine proteinase fractions produced a disruption of thenormally contiguous peripheral staining pattern of the TJ protein ZO-1and of the punctate staining of desmoplakin. Addition of E-64 to thecysteine proteinase fraction or AEBSF to the serine proteinase fractioninhibited the proteinase-dependent changes (FIG. 5).

HDM Proteinases Induce Cell Death in Epithelia

Control incubation of MDCK or Calu-3 cell monolayers for 18 h inserum-free EMEM produced negligible release of LDH until they weresubjected to hypotonic lysis at the end of the experiment (FIG. 6).Treatment of monolayers of either cell type with the cysteine and serineproteinase fractions also failed to release significant amounts of LDHinto the incubation medium (FIG. 6), despite the fact that some cellsdetached from the matrix substratum during the experiment. Lysis of thedetached cells and the adherent cells resulted in the recovery of LDHequivalent in amount to that found in untreated cell lysates (FIG. 6).

Cell death was also studied by examining DNA fragmentation and studyingthe staining of cells with AV and PI. FIG. 7a,b shows evidence of DNAfragmentation in MDCK and Calu-3 cells following treatment with HDMproteinases under conditions that result in an increase in permeabilityof epithelial monolayers. The effects of both HDM proteinase fractionswere attenuated by the matrix metalloproteinase inhibitor BB-250 (FIG.7b). E-64 inhibited the effects of the cysteine proteinase fraction, butnot the serine proteinase fraction (FIG. 7b). FIG. 7 also shows thattreatment of MDCK or Calu-3 cells with the serine proteinase fractionresulted in some cells within monolayers binding annexin V alone(AV⁺PI⁻), indicative of early apoptosis in these cells, and others whichstained with both annexin V and PI (AV⁺PI⁺) indicating cell death (seeFIG. 7d,f,h,j). The spatial distribution of AV⁺PI⁻early apoptotic cellsand AV⁺PI⁺dead cells was clustered around regions where there was cleardisruption of cell adhesion (eg see FIG. 7d,f).

Discussion

HDM faecal pellet proteins are a major cause of allergic asthma (Toveyet al., 1981) and in large part underlie the increasing prevalence ofthe disease (Dowse et al., 1985). In this study we have shown thatproteinases from D. pteronyssinus faecal pellets exert potent biologicaleffects on epithelial cells. The HDM proteinases were fractionated byammonium sulphate precipitation into cysteine and serine classes. Bothprecipitates had similar effects in the experimental systemsinvestigated. They produced an increase in permeability of epithelialmonolayers, caused cleavage of lateral cell adhesion, and detached cellsfrom the biomatrix substratum. Cleavage and detachment of cells was notassociated with gross release of LDH, but evidence was found of earlyapoptosis and outright cell death with nuclear rupture. Inspection ofcells stained with AV and PI revealed that staining was localized toareas of cell disruption/detachment. We further demonstrated that celldeath could be attenuated by proteinase inhibitors.

Ammonium sulphate precipitation proved to be an effective means ofseparating the major classes of proteinase activity in the spent HDMculture medium. The fraction precipitated by 50% ammonium sulphate hadcysteine proteinase activity and its permeability promoting effect onepithelial cell monolayers was inhibited by E-64 but not the serineproteinase inhibitor AEBSF. SDS-PAGE and immunoblot analysis with mAb5H8 raised against the HDM allergen Der p1 (a cysteine proteinase)revealed the presence of bands with apparent molcular masses of ˜22 KDaand ˜38 KDa in this fraction. In contrast, the fraction precipitated by50-80% saturated ammonium sulphate had serine proteinase activity andits effects on epithelial cells were inhibited by AEBSF, and to a lesserdegree SBTI, but not at all by E-64. We conclude from the studies withinhibitors that there was a minimal functional carry over of thecysteine proteinase allergen into this fraction. SDS-PAGE and immunoblotanalysis revealed the presence of several protein bands in the serineproteinase fraction, some of which were recognized by mAb 5H8 (egapproximate apparent masses 22, 26, 28, 29 and 38 KDa). The mAb 5H8 iswidely used to purify and quantify Der p 1, but its specificity hasrecently been called into question (Cambra & Berrens, 1996). Thus, anincidental finding from our study is cause for further concern regardingthe specificity of this antibody. The protein bands detected in the50-80% ammonium sulphate precipitate are consistent with the presence ofthe serine proteinase allergens Der p 3, Der p 6 and Der p 9. On thebasis of these observations we suggest that ammonium sulphatefractionation of HDM culture extracts provides a simple and effectivemeans to study the biological effects of HDM proteinase allergens andalso to identify novel inhibitors of their effects.

We have previously shown that highly purified Der p 1 allergen inducesan increase in the transepithelial flux of serum albumin in the airwaymucosa, causes disruption of epithelial architecture and detaches MDCKcells from natural biopolymer substrata (Herbert et al., 1990; 1995). Wealso demonstrated that these effects of Der p 1 were a result of itscysteine proteinase activity because they were sensitive to inhibitionby E-64, a relatively specific inhibitor of most cysteine proteinases(Barrett et al., 1982; Shaw, 1994; Herbert et al., 1995). Althoughcomparisons of amino acid sequence predict that Der p1 is a putativecysteine proteinase (Chua et al., 1988; 1993; Stewart, 1994; Topham etal., 1994; Robinson et al., 1997), others have suggested that it mightact as a bifunctional cysteine-serine proteinase because its activityhas also been reported to be inhibited by APMSF, an inhibitor of serineproteinases (Hewitt et al., 1995, 1997). If correct, this proposedbifunctionality would have potentially important implications for thedesign of specific inhibitors of Der p 1. However, in the present studywe argue against the functional significance of claimed bifunctionalityby showing (i) that the cysteine proteinase fraction derived from HDMcultures could not be inhibited by concentrations of AEBSF thatsignificantly inhibited the activity of the serine proteinase fraction,and (ii) the serine proteinase fraction being resistant to inhibition byE-64 at concentrations at which this inhibitor blocks cysteineproteinase activity. Other evidence against Der p 1 being a mixedcysteine-serine proteinase has also been presented recently (Chambers etal., 1997).

The permselectivity of the bronchial epithelium to hydrophilic solutesis governed by TJs which are expressed circumferentially at the apicalpole of each cell (Schneeberger & Lynch, 1992). Contiguous expression ofthe TJ proteins of one cell and their close opposition with TJ proteinson adjacent cells is thought to result in the epithelium being able todevelop its tight properties (Anderson & Van Itallie, 1995; Robinson,1995). Disruption of the interaction of the TJ proteins between cells,for example by the formation of discontinuities in their perijunctionallocalization, is associated with failure of epithelial barriers (Howarthet al., 1994; Zhong et al., 1994; Stuart et al., 1994 Stuart & Nigam,1995). Both fractions of HDM proteinase used in this study causedbreakdown of TJs as assessed by loss of perijunctional staining of ZO-1.Some disruption of desmosomes was also observed. The breakdown of TJsresulted in an increased permeability of epithelial monolayers, andeventually physical detachment of cells from the substratum occurred.The loss of ZO-1 from TJ and desmoplakin from desmosomes was dependentupon exogenous proteinase activity because the process was attentuatedby E-64 (in the case of the cysteine proteinase fraction) and AEBSF (inthe case of the serine proteinase fraction). Although ZO-1 anddesmoplakin are intracellular proteins, and thus unlikely to be degradedby exogenous proteinases, their breakdown is explicable as a consequenceof disruption of other, membrane-exposed, components of TJ anddesmosomes. A similar mechanism has been invoked to account for changesin other intracellular proteins following cleavage of intercellularcontacts (Volk et al., 1990).

The effects of the proteinases did not result in significant release ofLDH by the cells. However, treatment with either of the proteinasefractions resulted in some cells exhibiting signs of early apoptosis(AV⁺PI⁻) or outright cell death (AV⁺PI⁺). Cells may enter apoptosis bymultiple mechanisms (reviewed in Hale et al., 1996) including changes inhomotypic and heterotypic cell adhesion and cell-matrix attachment(Boudreau et al., 1995,1996; Mahida et al., 1996; Frisch & Francis,1994). An early signalling event in programmed cell death is disruptionof phospholipid binding cytoskeletal proteins which leads to thetransmembrane redistribution of phosphatidylserine (Martin et al.,1995a,b). The framework of cytoskeletal proteins is normally stabilizedand restrained by direct interaction with protein components ofintercellular junctions (Furuse et al., 1994; Anderson & Van Itallie,1995) which suggests that proteolysis of intercellular adhesions,especially TJ, could be the critical event in orchestrating the cellularresponse to proteinase allergens. The ability of E-64 to inhibit theaction of the cysteine but not serine proteinase fraction suggests thatE-64 acted at a proximal step in the process leading to permeabilitychanges and apoptosis, rather than by inhibiting a distal proteolyticstep in a transduction mechanism. Furthermore, intracellular signallingproteinases of the ICE/ced-3 caspase family that are activated interalia by Fas/APO-1 ligation in apoptosis (Los et al., 1995; Mariani etal., 1995; Kayagaki et al., 1995; Tanaka et al., 1996) have an unusualinhibitor profile in being insensitive to E-64. In contrast, theinhibitory action of BB-250 may occur through prevention of Fas ligandrelease (Mariani et al., 1995; Kayagaki et al., 1995).

Lung sensitization to airborne allergens such as those of HDM is centralto the pathogenesis of allergic asthma. The lung epithelium forms abarrier that foreign proteins must cross before they can cause allergicsensitization, but the mechanism by which allergens cross the epithelialbarrier is poorly understood (Robinson et al., 1997). The enzymaticnature of proteins derived from HDM feacal pellets provides oneexpalantion of the mechanism by which allergens encounter the immunesystem. By causing focal disruption of tight junctions, and ultimatelythe loss of a moribund cell, HDM proteinases would be able to increasethe paracellular permeation of allergens to antigen presenting cells. Itis noteworthy that the localized trauma and cell death produced byproteolytic cleavage of intercellular junctions may also fulfill someconditions of the ‘danger model’ of adaptive immunological response andthus explain why antibody-directed responses are evoked (Matzinger,1994; Ridge et al., 1996). In further support of this view recentevidence has suggested that when apoptosis (often considered to be animmunologically ‘silent’ form of cell death) occurs in the presence oftissue injury the resulting combined stimulus is actually threatening toantigen presenting cells (Boockvar et al., 1994; Casciola-Rosen et al.,1994; Ibrahim et al., 1996).

In summary these results show that HDM proteinases have effects onepithelial cells which are likely to promote allergic sensitization. Theability of specific inhibitors to interfere with epithelial cellresponses to proteinase allergens provides a rationale for theprevention and/or treatment of allergic conditions.

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LEGENDS TO FIGURES

FIG. 1.

SDS-PAGE (panel a) and immunoblot analysis (panel b) of HDM proteinasefractions. Key to lanes in panel a: immunoaffinity purified Der p 1 (1);HDM cysteine proteinase fraction (2) and HDM serine proteinase fraction(3). Proteins were visualized by coomassie blue staining. Panel b showsthe immunoblot prepared from the gel in panel a. Proteins were detectedusing mAb 5H8 (anti-Der p 1) and visualized by ECL technique. Note thedetection of immunoreactive proteins by mAb 5H8 in the serine proteinasefraction in addition to the expected immunoreactivity of the cysteineproteinase fraction. Filled circles indicate mass calibration standardsand arrows indicate apparent molecular masses of bands calculated fromthe mobilities of dye labelled standards.

FIG. 2.

Dilution-response curves showing the effects of 18 h exposure of cellmonolayers to the serine proteinase fraction prepared from HDM cultures.Panel (a) shows the mannitol permeability of MDCK epithelial cells undercontrol conditions (serum-free EMEM alone) and following treatment(proteinase fraction diluted in serum-free EMEM). Panel (b) depictssimilar studies performed in Calu-3 bronchial epithelial cells. Data aremean ± s.e. mean in 4-6 experiments. Proteinase allergen activity(expressed in azocoll units ml⁻¹) is indicated by the numbers under thefilled bars. Asterisks indicate statistically significant differenceswith respect to the untreated control response (serum-free EMEM medium)for each cell type .

FIG. 3.

Inhibition of the effects of HDM proteinase fractions on mannitolpermeability in MDCK cell monolayers. Panel (a) shows the effects ofE-64 (10μM) on the effect produced by the cysteine proteinase fraction(80 azocoll units ml⁻¹). Control cells were exposed to serum-free EMEMcontaining 0.5 mM reduced glutathione. Panel (b) depicts the effcts ofAEBSF (100μM) on the effects of the serine proteinase fraction (127azocoll units ml⁻¹). Data are mean i s.e. mean in 3-5 experiments.Contact time was 18 h in all cases. In both panels (a) and (b)statistically significant differences exist between the permeabilitiesof control and proteinase treated monolayers and also between themonolayers treated with the proteinases in the presence and absence ofinhibitors. Significant comparisons are shown in the Figure by thebracketing lines and indicated probability values.

FIG. 4.

Panel (a) illustrates the effects of 100μM AEBSF on the changes inmannitol permeability evoked in Calu-3 cell monolayers following 18 hexposure to the cysteine proteinase fraction (80 azocoll units ml⁻¹).Data are mean ± s.e. mean in 3 experiments. Panel (b) illustrates theeffects of 10μM E-64 on the changes in mannitol permeability evoked inCalu-3 cell monolayers following 18 h exposure to the serine proteinasefraction (94 azocoll units ml⁻¹). Data are mean ± s.e. mean in 3experiments. In both examples, the control cells were treated withserum-free EMEM medium alone (with 0.5 mM reduced glutathione in thecase of the cysteine proteinase fraction).

FIG. 5. Immunostaining of the TJ protein ZO-1 (panels a-e) and thedesmosomal plaque protein desmoplakin (panels f-j) in MDCK cellmonolayers. Panels a,f show immunostaining of cells exposed toserum-free EMEM as control. The pattern of immunostaining aftertreatment with the cysteine proteinase fraction (80 azocoll units ml⁻¹)is shown in panels b,g and the modification of this response by E-64(10μM) in panels c,h. The pattern of immunostaining after treatment withthe serine proteinase fraction (94 azocoll units ml⁻¹) is shown inpanels d,i and the modification of this response by AEBSF (100μM) inpanels e,j.

FIG. 6.

Measurement of LDH release following treatment of MDCK or Calu-3 cellswith HDM proteinase fractions for 18h. Panel a shows the lack of releaseof LDH from cells under control conditions until the monolayer wassubjected to hypotonic lysis. Panel a also shows that treatment with thecysteine proteinase fraction (80 azocoll units ml⁻¹ after activationwith 0.5 mM reduced glutathione) resulted in no release of LDH into themedium during the treatment period. Note that all of the cells weredetached from the wells during treatment and that hypotonic lysis of thedetached cells resulted in recovery of the same amount of LDH activitymeasured in the control cells. Panel b illustrates a similar experimentusing the serine proteinase fraction (114 azocoll units ml⁻¹). Note thatnot all of the cells were detached during the treatment period, but thatthe sum of LDH activity in lysed adherent and lysed detached cellscorrespond to the amount detected in control cells. Panels c and d showsimilar experiments performed in Calu-3 cells. Note that there was asmall background release of LDH from these cells (<10% total cellularLDH) under control conditions and that this was not significantlyaltered by proteinase treatment. Data shown are mean + s.e. mean in 4experiments.

FIG. 7.

Agarose gel electrophoresis of DNA (panels a,b) and cellular stainingwith AV and PI (panels c-j) following treatment of MDCK and Calu-3 cellswith HDM proteinase fractions. Panel a shows proteinase-induced DNAfragmentation in MDCK cells. Key to lanes: DNA markers (1,10); untreatedcells (2,3); cells treated for 18 h with 10μM camptothecin (positivecontrol) (4,5); cells treated for 18 h with serine proteinase fraction(114 azocoll units ml⁻¹) (6,7) and cells treated with cysteineproteinase fraction (80 azocoll units ml⁻¹ after activation) for 18 h(8,9). Panel b shows DNA fragmentation in Calu-3 cells. Key to lanes:DNA markers (1,11); untreated cells (2); cells treated for Xh withserine proteinase fraction (94 azocoll units ml⁻¹) (3); cells treatedwith cysteine proteinase fraction (80 azocoll units ml⁻¹ afteractivation) for 18 h (4); untreated cells in the presence of BB-250(5μM) (5); as lane 3, but cells treated in the presence of 5μM BB-250(6); as lane 4, but cells treated in the presence of 5μM BB-250 (7);untreated cells in the presence of 10μM E-64 (8); as lane 3, but cellstreated in the presence of 10μM E-64 (9); as lane 4, but cells treatedin the presence of 10μM E-64 (10). Panels c-f show fluorescencemicroscopy of AV and PI (e,f) staining of Calu-3 cell monolayers undercontrol conditions (c,e) and following treatment for 18 h with theserine proteinase fraction (135 azocoll units ml⁻¹) (d,f). Panels c,dshow the staining pattern under blue light excitation and e,f show thatunder green light excitation. Panels g-j show examples from MDCK cellmonolayers with the same layout as panels c-f. In both cell types, notethe virtual absence of AV and PI staining in untreated cells. In panelsd,f the staining pattern partially circumscibes an area of celldetachment in the monolayer and note that some cells exhibit both AV andPI staining. In panels h,j the majority of stained cells are positivefor both AV and PI and no significant detachment of cells from thesubstratum was evident.

What is claimed is:
 1. A method of treating a subject for the preventionor treatment of an allergic condition in which an allergen traverses anepithelial barrier comprising administering Lo the subject etherapeutically effective amount of an inhibitor of cysteine proteinaseactivity in conjunction with an inhibitor of serine proteinase activity.2. The method according to claim 1 wherein the allergeic condition isasthma.
 3. The method according to claim 1 wherein the allergiccondition is an allergic condition selected from rhinitis, allergicconjunctivitis, atopic dermatitis or food allergies.
 4. The methodaccording to claim 1 wherein the inhibitor of serine proteinase activityis not trypsin.
 5. The method according to claim 1 wherein the inhibitorof serine proteinase activity is an allergen serine proteinaseinhibitor.
 6. The method according to claim 5 wherein the allergenserine proteinase inhibitor inhibits Der p3, Der p6 or Der p9.
 7. Themethod according to claim 1 wherein said method comprises administeringE-64 (L-trans-epoxysuccinyl-leucylamido-(4-guanidines) butane) inconjunction with AEBSF (4-(2-amino ethyl)-benzenesulphonyl fluoridehydrochloride).